RIBOSOME CONCENTRATION CONTRIBUTES TO DISCRIMINATION AGAINST POLY(A)(-) MESSENGER-RNA DURING TRANSLATION INITIATION IN SACCHAROMYCES-CEREVISIAE

Inactivation of Saccharomyces cerevisiae poly(A) polymerase in a strai n bearing the temperature-sensitive lethal pap1-1 mutation results in the synthesis of poly(A)(-) mRNAs that initiate translation with surpr ising efficiency. Translation of poly(A)(-) mRNAs after polyadenylatio n shut-off might result from an increase in the ratio of ribosomes and associated translation factors to mRNA, caused by the inability of po ly(A)(-) mRNAs to accumulate to normal levels, To test this hypothesis , we used ribosomal subunit protein gene mutations to decrease either 40 or 60 S ribosomal subunit concentrations in strains carrying the pa p1-1 mutation, Polyadenylation shut-off in such cells results in a nea rly normal ratio of ribosomes to mRNA as revealed by polyribosome sedi mentation analysis. Ribonuclease protection and Northern blot analyses showed that a significant percentage of poly(A)-deficient and poly(A) (-) mRNA associate with smaller polyribosomes compared with cells with normal ribosome levels, Analysis of the ratio of poly(A)-deficient an d poly(A)(-) forms of a specific mRNA showed relatively more poly(A)- mRNA sedimenting with 20-60 S complexes than do poly(A)i forms, sugges ting a block in an early step of the translation initiation of the pol y(A)(-) transcripts. These findings support models featuring the poly( A) tail as an enhancer of translation and suggest that the full effect of a poly(A) tail on the initiation strength of a mRNA may require co mpetition for a limited number of free ribosomes or translation factor s.